Hepatitis C virus (HCV) is an important human pathogen that can cause severe liver diseases including liver cirrhosis and hepatocellular carcinoma. Recently, our laboratory discovered that HCV could perturb the autophagic pathway, leading to the accumulation of autophagosomes in cells. Autophagy plays an important role in maintaining cellular homeostasis. The ability of HCV to persistently perturb the autophagic pathway during chronic infection can have profound consequences in HCV pathogenesis and oncogenesis. Our further studies revealed the association of the HCV RNA replication complex with autophagosomal membranes, suggesting that HCV induces autophagosomes to facilitate its RNA synthesis. HCV apparently induces autophagosomes by inhibiting their fusion with lysosomes. Interestingly, our preliminary studies also indicate that HCV induces the biogenesis of autophagosomes via a novel mechanism independent of the class 3 phosphatidylinositol-3-kinase (PI3KC3). In this application, we propose to continue our novel findings to study how HCV inhibits the maturation of autophagosomes. Specifically, we will test the hypothesis that HCV induces Rubicon to sequester UVRAG from the HOPS complex to inhibit the maturation of autophagosomes. In addition, we will also elucidate the molecular pathway of HCV-induced biogenesis of autophagosomes. Finally, we will also use a novel approach that we recently developed to purify autophagosomes from HCV-infected cells and to characterize their associated protein factors to understand the biogenesis of these membrane vesicles and the relationship between autophagosomes and the HCV RNA replication complex. Our proposed research will generate important information for us to understand the interaction between HCV and its host cell and lead to a better understanding of HCV replication and pathogenesis. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is an important human pathogen. There are approximately 3.2 million people in the U.S. that are chronically infected by this virus. Many of these patients will develop severe liver diseases including liver cirrhosis and liver cancer and will require liver transplantation for survival. The goal of our proposed research is to understand the interaction between HCV and hepatocytes and how that interaction affects HCV replication and the progression of liver diseases. Our research will improve our knowledge about this important pathogen and lead to the improvements of treatments for HCV patients.